The present invention relates to stem cell compositions and methods for preparing them in vitro or ex vivo by culturing stem cells in medium containing anti-TGF-xcex2 antibodies in the absence of exogenously provided cytokines. Such treatment facilitates the survival of long term repopulating hematopoietic stem cells (LTR-HSC) within the culture for at least 14 days without replication or differentiation, the rapid engraftment of such LTR-HSC following in vivo administration to a mammal or the rapid proliferation of such LTR-HSC following transfer to in vitro culture conditions effective to result in such expansion.
The hematopoietic stem cell (HSC) is a pluripotent progenitor cell that has been characterized as a cell that is transplantable, can self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages.
Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, while differentiation of HSCs results in a loss of such multilineage potential, and corresponding lineage commitment with a progressive reduction of their clonal lifespan. Previous studies indicated that survival of HSC ex vivo in the absence of growth factors is limited, resulting in a complete loss of HSC after about 0.5-4 days in culture (Bartelmez S, unpublished data; Ploemacher R E et al. Stem Cells 11:336-347, 1993; Li and Johnson, Blood 15;84(2):408-14, 1994).
Transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodeficient mouse (Smith, L G et al., Proc Natl Acad Sci USA 88, 2788-92, 1991: Osawa M et al., Science 273, 242-245, 1996). In addition, repopulation of secondary (and tertiary) recipients, has been demonstrated (Dick J E et al Cell 42, 71-9, 1985; Jordan C T et al., Genes Dev 4, 220-32, 1990; Keller G and Snodgrass R J Exp Med 171, 1407-18, 1990).
Transforming growth factor beta-1 (TGF-xcex21) is known to directly and reversibly inhibit the initial cell divisions of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. (See, e.g., Sitnicka E et al, Blood, 88(1):82-88, 1996 and Ploemacher R E et al., Stem Cells 11(4):336-47, 1993.) The in vivo administration of TGF-xcex2 to humans to enhance the number of hematopoietic progenitor cells in peripheral blood has also been described. (See, e.g. U.S. Pat. No. 5,674,843, issued Oct. 7, 1997.) The mode of action of the observed pleiotrophic effect of TGF-xcex2 on stem and progenitor cells has been attributed to TGF-xcex2 as an inhibitor of cell proliferation or a mediator of apoptosis.
Murine marrow cells treated with anti TGF-xcex2 antibody together with IL-3, IL-6 and stem cell factor demonstrated a greater retroviral transduction efficiency of progenitor (CFU-C) and long-term repopulating cells than cells treated with IL-3, IL-6 and stem cell factor alone (Yu J et al., Gene Ther 5(9):1265-71, 1998).
Extensive studies have been described wherein HSC are cultured in the presence in various combinations of cytokines as a means to increase the number of HSC. In general, such culture conditions have caused differentiation of HSC and do not result in survival or increased numbers of viable long term repopulating HSC (Li and Johnson, Blood 15;84(2):408-14, 1994; Peters S O et al., Blood. 87(1):30-7. 1996; Yonemura Y et al., Proc Natl Acad Sci USA. 93(9):4040-4. 1996).
High-dose chemotherapy and/or radiation therapy together with bone marrow transplantation or transplantation of a cell population enriched for hematopoietic stem cells are standard treatment regimens for some malignancies, including, acute lymphocytic leukemia, chronic myelogenous leukemia, neuroblastoma, lymphoma, breast cancer, colon cancer, lung cancer and myelodysplastic syndrome, as well as for other non-malignant hematopoietic diseases, e.g. thrombocytopenia. Such treatments have shown promise in effective elimination of several types of cancer, however in all cases the high doses also destroy bone marrow stem cells. In addition, bone marrow transplantation may play a major role in the emerging field of gene therapy.
Clinical trials are underway using such regimens for the treatment of various cancers, including ovarian cancer, thymomas, germ cell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer, and brain tumors.
In addition, HSC have been demonstrated to be capable of repopulating non-hematopoietic tissues, including but not limited to liver (Petersen B E et al., Science 284:1168-70, 1999) and neuronal tissue (Bjornson C R R et al., Science 283:534-7, 1999).
Cell preparations enriched for hematopoietic stem cells generally contain a low percentage of cells capable of long-term hematopoietic reconstitution. In general, culture conditions effective to promote the survival of hematopoietic stem cells include cytokines, which stimulate cell division and differentiation of the cells, diminishing their long term repopulating capability. Frequently, as a result, in vivo administration of such cell preparations does not result in rapid repopulation of the host hematopoietic system. In particular, the slow repopulation of the neutrophil and platelet compartments of the hematopoietic system may result in susceptibility to infection and/or complications due to poor blood clotting. In addition, once isolated, stem cell preparations are typically frozen in liquid nitrogen for subsequent use and upon thawing the number of viable stem cells is further reduced.
Therefore, a need remains to develop techniques for maintaining stem cells in culture and for the use of such cells in both rapid and long-term hematopoietic reconstitution.
The present invention addresses two significant problems in the field of stem cell transplantation. As known in the art, hematopoietic stem cells capable of long term repopulation in vivo generally do not survive in culture without cell division, which usually results in differentiation of the cells out of the stem cell compartment. In addition, the time required for in vivo repopulation of the hematopoietic system of a subject following in vivo administration of such stem cells is sufficiently long that passive administration of platelets and neutrophils is often required to ensure the survival of the patient.
The present invention provides a composition of anti TGF-xcex2 antibody-treated stem cells capable of stivival for at least 14 days in vitro or ex vivo with continuous anti TGF-xcex2 antibody treatment and a method for obtaining the same.
The anti TGF-xcex2 antibody-treated stem cell compositions of the invention provide a source of stem cells for rapid and sustained repopulation of the hematopoietic system of the subject. The compositions of the present invention provide a number of advantages relative to currently available stem cell preparations including: (1) hematopoietic repopulation which takes place at least 2-fold more rapidly following in vivo administration; (2) hematopoietic repopulation in vivo with a minimal number of cells, e.g., at least 10-fold fewer stem cells if antibody-treated (3) sustained repopulation of the hematopoietic system of the subject for a clinically useful time; (4) stem cell proliferation in vitro which takes place at least 2-fold more rapidly following transfer to culture conditions effective to promote such proliferation; (5) stem cell proliferation in vitro with a minimal number of cells, e.g., at least 10-fold fewer stem cells if antibody-treated; and (6) sustained stem cell proliferation in vitro resulting in generation of various lineages of hematopoietic cells for at least six months.
In one preferred aspect of the invention, the stem cells are human hematopoietic stem cells, characterized as lacking the expression of lineage markers (lin-), and either (a) positive for cell surface expression of CD 34 and KDR and negative for cell surface expression of CD38 or (b) positive for cell surface expression of both CD 34 and Thy1.
In another aspect, the culture conditions effective to preserve the viability and differentiation state of said stem cells include culture medium which contains from about 0.5 to 100 xcexcg/ml of anti TGF-xcex2 antibody and lacks exogenously provided cytokines.
The invention further provides a method of obtaining a stem cell composition characterized by prolonged survival in culture which includes the steps of obtaining a population of cells enriched for stem cells and exposing the stem cells, ex vivo, to an anti TGF-xcex2 antibody, under culture conditions, and for a period of time, effective to preserve the viability and differentiation state of the cells.
A method for rapid in vivo repopulation of the hematopoietic system of a subject and a method for rapid proliferation of a stem culture in vitro are further provided by the invention. Such methods include the steps of obtaining a population of cells enriched for stem cells and exposing the stem cells, ex vivo, to an anti TGF-xcex2 antibody, under culture conditions, and for a period of time, effective to preserve the viability and differentiation state of the cells, followed by either (a) readministering the anti TGF-xcex2 antibody treated stem cells to the subject or (b) transferring the anti TGF-xcex2 antibody treated stem cells to culture conditions effective to result in the rapid proliferation of the cells, for in vivo and in vitro applications, respectively.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.